Phenotypic plasticity and intraspecific variability in desalination tolerance in European sea bass Dicentrarchus labrax

Facing environmental change, organisms may have to flee or to acclimate. Acclimation is based on the ability of organisms tomodify their phenotype, i.e. some of their biological traits (metabolism, behaviour, physiology) in order to survive in a given environment.This is called phenotypic plasticity. However, individuals of the same species may differ within their genotype and phenotypes. This isreferred as inter-individual or intra-individual variability. This variability can be found in the plasticity of organisms to respond to rapid orlong-term changes in their environment. The European sea bass Dicentrarchus labrax is a marine fish whose range extends from the northernEuropean coasts to the African sides and the Mediterranean sea. It undertakes seasonal migrations from the juvenile stage in lagoons andestuaries and sometimes even in rivers. It is therefore a plastic species in terms of osmoregulatory physiology, capable of withstanding a widerange of salinity: from 0 to 90 ppt. On the other hand, in the laboratory, a high variability in the osmoregulatory response in fresh water wasfound. The objectives of this thesis were to characterise the phenotypic plasticity and intraspecific variability of the Mediterranean sea bass inrelation to desalination.To do this, fish were transferred to fresh water and studied by different approaches. The first one allowed me to understand theeffects of desalination on respiratory metabolism and hypoxia tolerance. In fresh water, the respiratory capacities of sea bass differ fromthose of seawater with a higher metabolic rate, as well as a higher tolerance to hypoxia. In addition, the study of the transcripts revealsdifferent responses after two weeks and two months in fresh water. Individuals unable to tolerate fresh water were characterised by lowerbehavioural traits (velocity, displacement) and biochemical traits (osmotic pressure, blood chloride and sodium) than those of freshwatertolerant. The study of transcripts (ionic transporters and hormone receptors) revealed that intolerance to fresh water is partly due to aninability at the renal level to reabsorb ions and thus maintain the hydromineral balance. The study of the phenotypic variability ofMediterranean sea bass in fresh water has been repeated at different ages. It shows that tolerance/intolerance to fresh water is a stablephenomenon in terms of proportions (30% intolerant), but labile over time (it is not necessarily the same individuals who are tolerant orintolerant to fresh water following successive transfers). This suggests random, genetic and/or epigenetic regulatory mechanisms. Apreliminary study of DNA cysteine methylation concluded that freshwater transfer influences overall DNA methylation. The link betweenintraspecific variability related to freshwater tolerance and DNA methylation remains to be investigated.The last approach used was to study telomere dynamics (length and expression of the telomerase gene) as potential markers ofhypo-osmotic stress in sea bass. The method, now in development, does not indicate any effect of hypo-osmotic stress on telomere dynamics.Nevertheless, the response of intolerant sea bass to fresh water has yet to be confirmed by increasing the number of individuals. Thesepreliminary results suggest that transitions to hypo-osmotic environments do not cause more oxidative damage at the branchial level. Itwould be interesting to use this approach in the context of other environmental stressors and to explore other organs telomere dynamics.Face à un changement dans leur environnement, les organismes peuvent être amenés à fuir ou à s’acclimater. L’acclimatation repose sur la capacité des organismes à pouvoir modifier leur phénotype, c’est à dire certains de leur traits biologiques (métabolisme,comportement, physiologie) afin de pouvoir survivre dans un environnement donné. On parle alors de plasticité phénotypique. Cependant,les autres individus d’une même espèce peuvent présenter des différences au sein de leur génotype et phénotypes. On parle alors de variabilité inter-individuelle ou intraspécifique. Cette variabilité peut se retrouver dans la plasticité que possèdent les organismes à répondre à des changements rapides ou à long terme de leur environnement. Le bar ou loup Européen Dicentrarchus labrax est un poisson marin dont l’aire de répartition s’étend des côtes nord européennes jusqu’aux côtés africaines en passant par la Méditerranée. Il entreprend des migrations saisonnières dès le stade juvénile dans les lagunes et les estuaires voire parfois même en rivière. C’est donc une espèce plastique en termes de physiologie osmorégulatrice, capable de supporter une grande gamme de salinité : de 0 à 90 ppt. En revanche, en laboratoire une forte variabilité dans la réponse osmorégulatrice en eau douce a pu être mise en évidence. Les objectifs de cette thèse étaient de caractériser la plasticité phénotypique et la variabilité intraspécifique du loup méditerranéen en lien avec la dessalure.Pour cela, des loups ont été transférés en eau douce et étudiés par différentes approches. La première m’a permis d’appréhender les effets de la dessalure sur le métabolisme respiratoire et la tolérance à l’hypoxie. En eau douce, les capacités respiratoires des loups diffèrent de celles en eau de mer avec un taux métabolique plus haut, ainsi qu’une tolérance à l’hypoxie plus accrue. De plus, l’étude des transcrits révèle des réponses différentes après deux semaines et après deux mois passés en eau douce. Les individus incapables de tolérer l’eau douce ont été caractérisés par des traits comportementaux (vitesse, déplacement) et biochimiques (pression osmotique,chlorure et sodium sanguins) plus faibles que ceux des tolérants à la dessalure. L’étude des transcrits (transporteurs ioniques et récepteurs aux hormones) a révélé que l’intolérance à l’eau douce serait due en partie à une incapacité au niveau rénale à réabsorber les ions et donc à maintenir leur balance hydrominérale. L’étude de la variabilité phénotypique des loups méditerranéens en eau douce a été répétée à des âges différents. Elle démontre que la tolérance/l’intolérance à l’eau douce est un phénomène stable en termes de proportions (30% d’intolérants),mais labile avec le temps (ce ne sont pas forcément les mêmes individus qui sont tolérants ou intolérants à la dessalure suite à des transferts successifs). Cela suggère des mécanismes de régulation aléatoire, génétiques et/ou épigénétiques. Une étude préliminaire de la méthylation des cystéines de l’ADN a permis de déduire que le transfert en eau douce influe sur la méthylation globale de l’ADN. Le lien entre variabilitéintra spécifique liée à la tolérance à l’eau douce et méthylation de l’ADN reste à étudier.La dernière approche menée a consisté à étudier la dynamique des télomères (longueur et expression du gène de la télomérase)comme potentiels marqueurs de stress hypo-osmotiques chez le loup. La méthode, désormais au point n’indique aucun effet du stress hypoosmotique sur la dynamique des télomères. Néanmoins, la réponse des loups intolérants à l’eau douce reste à confirmer en augmentant le nombre d’individus. Ces résultats préliminaires suggèrent que les transitions vers les environnements hypo-osmotiques ne provoquent pas plus de dommages oxydatifs au niveau branchial. Il serait intéressant d’aborder l’approche de la dynamique des télomères dans le cadre d’autres stress environnementaux et de creuser l’approche sur d’autres organes

The role of salinity on genome‐wide DNA methylation dynamics in European sea bass gills

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change

Intraspecific variation in freshwater tolerance has consequences for telomere dynamics in the euryhaline teleost Dicentrarchus labrax

International audienceStressful events can alter organism physiology at several levels triggering allostatic responses. Telomeres are well-conserved repetitive DNA sequences mainly localised at chromosome's ends, playing a crucial role in DNA stability. Analyses of telomere dynamics are new tools to assess consequences of environmental stress in non-model organisms like fish. In this study, the relationship between freshwater tolerance and telomere dynamics was investigated in the gills of the European sea bass Dicentrarchus labrax. Fluorescent in situ hybridisation of telomeric sequences revealed distal telomeres as well as intrachromosomal telomeres known as interstitial telomere sequences. In order to better understand telomere dynamics in the gills of D. labrax, we used quantitative PCR to measure telomere length and mRNA expression of the catalytic subunit of telomerase reverse transcriptase tert. For the calculation of the relative telomere length, two reference genes were tested: the single copy gene mc2r, encoding melanocortin 2 receptor and the multicopy gene 18S, encoding the 18S ribosomal RNA. We proposed a novel normalisation method to calculate the relative telomere length using both, single and multiple copy genes as references. Cell dynamics was also investigated by measuring mRNA expression of genes involved in apoptosis (caspase 8 and 9), cell proliferation (proliferation cell nuclear antigen), aerobic mitochondrial metabolism (ATP citrate-synthase), anaerobic metabolism (lactate dehydrogenase a) and antioxidant enzymatic defences (superoxide dismutase 1 and 2, catalase). Following a 15-days fresh water exposure, telomere dynamics was not significantly modified in the gills of freshwater tolerant fish. But freshwater intolerant fish exhibited telomere attrition relative to saltwater controls, and lower expression of tert in gills relative to freshwater tolerant fish. This modification of telomere dynamics in intolerant individuals was found to be correlated with lower antioxidant enzymatic defences, a higher aerobic metabolic marker and a lower cellular turnover. These data bring new perspectives for the use of telomere dynamics as an integrative marker to study environmental stress in fish, while considering individual phenotypic plasticity in response to freshwater exposure